EA011880B1 - Telomelysin-gfp gene-containing recombinant virus - Google Patents

Abstract

The present invention relates to provide a regent for detecting cancer cells or diagnosing cancer. More particular, said invention relates to providing a reagent for detecting cancer cells containing a recombinant virus in which a replication cassette having a human telomerase promoter, E1A gene, IRES sequence and E1B gene in this order is integrated into the E1 region of the virus genome while a labeling cassette having a gene encoding a labeled protein and a promoter capable of regulating the expression of the gene is integrated into the E3 region of the virus genome.

Description

Field of Invention

The present invention relates to a reagent for detecting cancer cells or to diagnosing cancer and a factor inducing cell death.

Known level of research

Telomerase activity is often increased in cells that underwent malignant transformation or in immortalized cell lines, and telomerase activity is difficult to determine in normal somatic cells, with the exception of germ line cells, blood cell lines and epithelial cells. Therefore, there have been attempts to identify cancer on the basis of telomerase activity as an indicator (8Nau IV., Ζου Υ., Nuasha E., Ugsh1I ν.Ε. Te1oshega8e ap Sapseg. Nit Mo1 Ssps1 10 (7): 677-85, 2001).

On the other hand, the detection of cancerous tissues and metastatic lymph nodes ίη νίνο has been intensively studied in the field of diagnostic imaging. For example, a biological analysis using PET and imaging analysis was reported, making full use of the neural network. Further, studies of the antitumor activity and safety of viruses with selective replication were reported (^ eVe8e T.I., νаη её Рё1 N., S 8., M1kNak V., Oge \ u K., Soeschapp M., Natreg I., Ekepd K., Oe1ope Ν., Koipdieh K., Nai1k T., OeMagho A.M., P1ap1ayo81 8., О.υ O.S., SNep Υ., Nepyegkop Ό.Κ., Sagyisa MA, No. 1cop \ \ '. S., 81shop IV. And pHake I 1a1o! SU706, And gerNsayop-soshrekp! 61 (20): 7464-72, 2001.) The inventors of the present invention also found to infect cancer cells of vi A rusome possessing a telomerase promoter and the ability to replicate can kill cancer cells by replication of the virus (Ka ^ akysha T., Kadara 8., Kauauki Ν., 8 гgak1ua Υ., Isheoka T., Tegaki E., Tact M., Kuo 8 ., Tapaka Ν., Apy Eitztaga T., Ke1a1ei Agyskk, bshkk Akkas! Tekshegake-kresyk herysaiopkeks Tsue uyoFegaru Gog Nishap sapseg, S1sh Sapseg Kek 10 (1): 285-92, 2004).

However, a system for detecting shyy cancer in surgical operations has not yet been developed due to the difficulties of targeting cancer cells. Further, to date, no studies are known in which the body would become infected with the virus and the kinetics of the virus inside the cancer cells would actually be used to visualize the cancerous tissues.

OBJECTS OF THE INVENTION

As indicated above, the desired goal was to develop a reagent to detect cancer cells or to diagnose cancer and the factor that causes cell death, and each reagent would be able to visualize cancer cells even w νί \ Ό.

As a result of intensive and extensive studies aimed at solving the above problems, the authors of the present invention found that it is possible to detect cancer cells at an extremely high sensitivity level and even w νί \ Ό by integrating a gene including fluorescence-labeled protein into the E3 region of the virus genome and integrating replication cassettes with the human telomerase promoter, the E1A gene, the 1KE8 sequence and the E1B gene in the named order to the E1 region, followed by the expression of both cassettes. Thus, the objective of the present invention has been achieved.

The essence of the present invention can be seen from the following description.

(1) A reagent for detecting cancer cells, including a recombinant virus, in which a replication cassette comprising a human telomerase promoter, the E1A gene, the 1KE8 sequence and the E1 B gene in the above order is integrated into the E1 region of the virus genome and a labeling cassette including the gene, a coding marking protein and a promoter capable of regulating the expression of a gene encoding a marking protein is integrated into the E3 region of the virus genome.

(2) A reagent for the diagnosis of cancer, comprising a recombinant virus, in which a replication cassette with a human telomerase promoter, E1A gene, 1KE8 sequence and E1B gene is integrated in the named order into the E1 region of the virus genome and a labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of a gene encoding a labeling protein is integrated into the E3 region of the virus genome.

According to the above paragraphs. (1) and (2), these reagents can be used to detect w νίνΌ, diagnose and navigational surgery. As a specific example, a human telomerase promoter, NTECT can be indicated. As a specific example of a marking protein, SER can be named. As a promoter capable of regulating the expression of a gene encoding a marking protein, for example, a cytomegalovirus promoter or NTECT-promoter can be used. As a virus, for example, adenovirus can be used.

(3) A cell death factor, including a recombinant virus, in which a replication cassette with a human telomerase promoter, the E1A gene, the 1KE8 sequence and the E1 B gene are included in the named order in the E1 region of the virus genome, as well as the cell death cassette, with a gene encoding a protein associated with the induction of cell death, and a promoter capable of regulating the expression of a gene encoding a protein associated with the induction of cell death, integrated into the E3 region of the virus genome.

In this factor inducing cell death, the promoter from human telomerase can be

- 1 011,880 iTEKT promoter. Examples of proteins associated with the induction of cell death include immunity-associated proteins, apoptosis inducing proteins and telomerase-associated proteins. More precisely, PA28 can be called as an immunity-associated protein, TK1b can be called as an apoptosis inducing protein, and LI5 can be called as a telomerase-associated protein. A promoter capable of regulating the expression of a protein associated with the induction of cell death may be a cytomegalovirus promoter or an iTEKT promoter; the virus may be an adenovirus. The cell of the present invention may be a cancer cell.

(4) A method for detecting cancer cells, comprising infecting the cancer cells with a reagent according to the above (1) and determining the fluorescence emitted by the cancer cells.

(5) A method for diagnosing cancer, including infection of cancer cells with a reagent according to the above (2) and determining the fluorescence emitted by the cancer cells.

(6) A method for inducing cell death of a target cell, comprising infecting the target cell with a factor inducing cell death according to the above (3).

Brief Description of the Figures

FIG. 1 is a diagram depicting the structure of telomelysin-SBR.

FIG. 2 is a diagram depicting replication of a non-replicating virus.

FIG. 3 is a diagram depicting the results of detecting cancer cells by co-infection with Ly-FBR ίη νίίτο.

FIG. 4 is a diagram depicting the results of detecting human cancer tissues by co-infection with Ly-FBR ίη νίνο.

FIG. 5 is a diagram depicting morphological changes in human lung cancer cells upon infection with telomelysin-RRF.

FIG. 6 is a diagram depicting RBF-fluorescence of human lung cancer cells infected with telomelysin-RBF.

FIG. 7 is a diagram depicting telomelysin-SBR replication determined by quantitative CSC (polymerase chain reaction) in real time.

FIG. 8 is a diagram illustrating RBF-fluorescence of human colon cancer cells infected with telomelysin-RBF.

FIG. 9 is a diagram depicting telomelysin-SBR replication determined by quantitative CSC (polymerase chain reaction) in real time.

FIG. 10 is a diagram depicting morphological changes in normal human lung fibroblast cells (ANL) upon infection with telomelysin-RRF.

FIG. 11 is a drawing depicting RBF-fluorescence of normal human lung fibroblast cells (PNBF) upon infection with telomelysin-RBF.

FIG. 12 is a diagram depicting a comparison of telomelysin-SBR replications determined by real-time quantitative CSC (polymerase chain reaction).

FIG. 13 is a diagram depicting intratumoral proliferation / replication of telomelysin-SBR observed by fluorescence imaging.

FIG. 14 is a diagram depicting intratumoral proliferation / replication of telomelysin-SBR observed by fluorescence imaging.

FIG. 15 is a diagram depicting intratumoral proliferation / replication of telomelysin-SBR in a lymph node metastasis model observed by fluorescence imaging.

FIG. 16 is a diagram showing a histological analysis of an orthotopic colorectal cancer model on hairless mouse and human colon cancer cancer cells HT29.

FIG. 17 is a diagram showing autopsy results on a model of orthotopic rectal cancer in a hairless mouse and HT29 human colon cancer cells.

FIG. 18 is a diagram depicting intratumoral proliferation / replication of telomelysin-SBR in a rectal HT29 tumor and near-aortic lymph nodes, observed by fluorescence imaging.

FIG. 19 is a diagram depicting intratumoral proliferation / replication of telomelysin-SBR in periaortic lymph nodes, observed by fluorescence imaging.

FIG. 20 is a diagram depicting intratumoral proliferation / replication of telomelysin-SBR in paraaortic lymph nodes, observed by fluorescence imaging.

Preferred Embodiment

The following is a detailed description of the present invention.

References cited in this specification are incorporated by reference in their entirety.

1. A reagent for detecting cancer cells and a detection method.

The present invention relates to a reagent for detecting cancer cells, including recombinant

- 2011880 binate virus, in which a replication cassette comprising a human telomerase promoter, E1A gene, sequence ΙΚΕ8 and E1B gene in the named order is integrated into the E1 region of the virus genome and a labeling cassette comprising a gene encoding a marking protein and a promoter capable of regulating expression of the gene encoding the marking protein is integrated into the E3 region of the virus genome. Further, the present invention relates to a method for detecting cancer cells, comprising infecting cancer cells with a given reagent and detecting fluorescence emitted by cancer cells. In the present invention, the term “recombinant virus” means a virus in which the replication cassette and the tag cassette described below are integrated into the genome. The virus used in the present invention is not strictly limited, but adenovirus is preferred from a security point of view. Among adenoviruses, type 5 adenovirus is particularly preferred since it is easier to work with.

The recombinant virus used in the present invention has a replication cassette integrated in the region corresponding to the E1 region of the adenovirus genome and a labeling cassette integrated in the region corresponding to the E1 region of the adenovirus genome. The replication cassette includes the human telomerase promoter, the E1A gene, the ΙΚΕ8 sequence, and the E1B gene in the named order. The E1A gene, the ΙΚΕ8 sequence, and the E1B gene are activated by the human telomerase promoter, which leads to cancer cell-specific and telomerase-specific virus proliferation / replication. A tagging cassette includes a promoter and a gene encoding a tagging protein. For example, a gene encoding a labeling protein is induced by the cytomegalovirus (SMU) promoter or ΗΤΕΚΤ-promoter (Fig. 1).

The “telomerase promoter” defines a transcription initiation site for telomerase and directly controls the frequency of transcription. Telomerase is an enzyme that maintains the length of telomeres, prevents the shortening of telomeres during the replication of eukaryotic chromosomes. The type of such a telomerase promoter is not particularly limited, and any suitable telomerase promoter compatible with the virus selected for expression of the gene of interest to us can be used. For example, a promoter for human transcriptase reverse transcriptase is preferred (11ΤΕΡΤ). A number of sequences that bind the transcription factor were confirmed in the region of 1.4 kbp in the opposite direction from the end of the 5 'gene 11ΤΕΡΤ. It is believed that this region is the 11ΤΕΡΤ promoter. In particular, the 181 Lp sequence (bp - base pairs), located in the opposite direction from the transcription initiation site, is the core region important for the expression of the gene located in the forward direction. Any sequence including this core region can be used in the present invention. It is preferable to use as an ΗΤΕΚΤ-promoter a reverse sequence of about 378 bp containing the entire core region. It has been proven that this sequence of approximately 378 bp equivalent to 181 bp core region by gene expression efficiency. The nucleotide sequence of the 11ΤΕΡΤ promoter of 455 bp shown in 8ΕΟ W N0: 1.

The nucleotide sequence of the ΗΤΕΚΤ promoter is not limited to the sequence shown in 8Ε0 ΙΌ N0: 1. The sequence of nucleotides that hybridize under stringent conditions into a DNA consisting of a nucleotide sequence complementary to DNA consisting of 8Ε0 ΙΌ N0: 1 and having ΗΤΕΚΤ promoter activity , can also be included as a sequence for the ΗΤΕΚΤ promoter. Such nucleotides can be obtained from cDNA libraries or genome libraries by such well-known hybridization methods as colony hybridization, plaque hybridization, Southern blotting, etc., using polynucleotides consisting of the 8Ε0 ΙΌ N0: 1 nucleotide sequence or its part as a probe. The cDNA libraries can be prepared according to the method described in Molecular Clibid: A baaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (Co1b 8ppp§ Nagog Przegz (1989)). Another possibility is the use of commercial cDNA libraries or genome libraries.

Under the above hybridization, examples of stringent conditions include 1x88C - 2x88C, 0.1-0.5% 8E8 and 42-68 ° C. More specifically, you can give an example in which prehybridization is performed at 60-68 ° C for more than 30 minutes, and then washing is performed in 2x88C, 0.1% 8-8 at room temperature for 5-15 minutes 4-6 times.

Detailed descriptions of the hybridization procedures can be found, for example, in Moiociulagus clusidus: Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. (Co1b 8rgd NagGr Prozs (1989)), especially in sections 9.47–9.58.

The reason why the E1A gene, the ΙΚΕ8 sequence, and the E1B gene are arranged in this order in the present invention is because the insertion of the ΙΚΕ8 sequence between the E1A gene and the E1 B gene gives an increased ability to replicate the virus when the host cell is infected with it. The E1A gene and the E1B gene are genes that make up the E1 gene. This is one of the earliest virus genes that have early (E) genes and late (b) genes, which takes part in the replication of their DNA and encodes a protein involved in the transcription of the virus genome. Protein E1A, encoded by the Ε1Α gene, activates the transcription of a group of genes (Е1В, Е2, Е4, е! С.), Which are necessary to obtain an infectious virus. The E1B protein encoded by the E1B gene promotes the accumulation of the late tDNA gene (b gene) in the cytoplasm of the infected host cell and inhibits protein synthesis in the host cell. That

- 3 011880 thus, the E1B protein promotes the replication of the virus. The nucleotide sequences of the E1A gene and the E1B gene are shown in BEO ΙΌ N0: 2 and BEO GO N0: 3, respectively.

E1A and E1B can have, in addition to the nucleotide sequences shown in BEO GO N0: 2 and BEO GO N0: 3, respectively, nucleotide sequences that hybridize under stringent conditions to form OH. consisting of BEO GO N0: 2 or BEO GO N0: 3, and encode a protein with E1A or E1B activity. Such nucleotide sequences can be obtained from cDNA libraries or genome libraries based on known methods, such as colony hybridization, plaque hybridization, Southern blotting, etc., using a polynucleotide or part of it consisting of the nucleotide sequence BEO GO N0: 2 or BEO GO N0: 3 as a probe, with DNA, can be obtained by the methods described in MoIci1ag Closid: A baoba ogu Mapia1 2ib eb. (Co1B Brpid Nagog Przeg (1989)). Or it is possible to use commercial cDNA libraries or genome libraries. Under the above hybridization, stringent conditions include 1xBBS - 2xBBS, 0.1-0.5% BIB and 4268 ° C. More specifically, you can give an example in which prehybridization is performed at 60-68 ° C for more than 30 minutes, and then washing is performed in 2xBBS, 0.1% BIB at room temperature for 5-15 minutes 4-6 times.

Detailed descriptions of the hybridization procedures can be found, for example, in Moiociulagus clusidus: Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. (Co1b Brpid Nagog Przegz (1989)), especially in sections 9.47–9.58.

ΙΒΕ8 (1i1egia1 bosote Ei! Gu Bie - the internal site of entry into the ribosome) is a signal for the initiation of protein synthesis specific for picornavirus. The site is believed to serve as a ribosome binding site because it has an additional sequence to the 3 ′ terminal sequence of 188 ribosome DNA. It was shown that tDNA obtained from the virus belonging to rkotautbay is transmitted through this sequence. Translational efficiency through the HBVE sequence is high. Even from the middle of tDNA, protein synthesis occurs regardless of the cap structure. Therefore, in the virus of the present invention, both the E1A gene and the E1B gene (which is located in the forward direction from the HBEB sequence) are independently transmitted by the human telomerase promoter. With the use of GVEB, expression is controlled by the telomerase promoter independently on the E1A gene and E1 B gene. Therefore, in comparison with cases where either the E1 A or E1 B gene is controlled by the telomerase promoter, virus replication can be more strictly confined to cells with telomerase activity . The sequence of GVEB is shown in BEO GO N0: 4.

GVEB can have, in contrast to the nucleotide sequences shown in BEO GO N0: 4, nucleotide sequences that hybridize under stringent conditions in DNA, consisting of the nucleotide sequence BEO GO N0: 4, and encode a protein with HBE activity. Such nucleotide sequences can be obtained from cDNA libraries or genome libraries by such well-known hybridization methods as colony hybridization, plaque hybridization, southern blotting, etc., using polynucleotides consisting of the BEO GO N0: 1 nucleotide sequence or part thereof as a probe. The cDNA libraries can be prepared according to the method described in MoIci1ag C1iSid: Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (Co1B Brpid Nagog Przeg (1989)). Another possibility is the use of commercial cDNA libraries or genome libraries. Under the above hybridization, examples of severe conditions include 1xBBS - 2xBBS, 0.1-0.5% BIB and 42-68 ° C. More specifically, you can give an example in which prehybridization is performed at 60-68 ° C for more than 30 minutes, and then washing is performed in 2xBBS, 0.1% BIB at room temperature for 5-15 minutes 4-6 times. Detailed descriptions of the hybridization procedures can be found, for example, in Moiociulagus clusidus: Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. (Co1b Brpid Nagog Przegz (1989)), especially in sections 9.47–9.58.

In the present invention, the promoter from human telomerase is located in the opposite direction from the E1 gene, since such a promoter can stimulate the replication of cells with telomerase activity.

Genes containing replication cassettes of the present invention can be obtained using conventional genetic engineering methods. For example, as a genetic engineering method, a nucleic acid synthesis method with a commonly used DNA synthesizer can be used. Or, after isolating or synthesizing the genetic sequence that you intend to use in the form of a matrix, primers specific for each gene can be developed, and the genetic sequence can be amplified using the RSV apparatus (RSV method; SiggyGrr! ^ Yeu & Boise (1987), Besoi 6.1-6.4); or a gene amplification method using a cloning vector can be used. With ordinary skills in this area, it is easy to carry out the methods mentioned above using, for example, the guidance of Moibeciag Clioid 2 ^ib Eb., Cob Brpid Nagog Lobogogo Przez (1989). Purification of the obtained RSV products can be carried out by known methods, such as the method with ethidium bromide, the method with green BWB Ι (molecular probes), the method with SECHESELKh (Eiiakozi), OMCEX (O ^ SEX), etc., using an agarose gel, the method using IEAE-cellulose filter, freeze-squeeze method or dialysis tube method. When an agarose gel is used, the PCT products are subjected to agarose gel electrophoresis and the resulting DNA fragments are excised from the gel and purified. If necessary, it is possible to confirm the usual

- 4 011880 using sequencing methods to obtain the expected gene. For example, for this purpose, you can use the method of chain dideoxynucleotide termination (8apdeg c1 a1. (1977) Proc. No. 111. Asab. 8c1. I8A 74: 5463) or similar methods. Another possibility is that you can also analyze the sequence with a suitable DNA sequencer (for example, AB1 PK18M; Arrieb Wyuzuzetz).

Further, the obtained individual genes are ligated in a specific order. First, the above genes are treated with known restriction enzymes and the resulting DNA fragments are introduced into a known vector, in accordance with a known ligation method. Examples of known vectors include the p1PE8 vector including ΙΡΕ8 (the site of the internal entrance to the ribosome in tDNA) of the encephalomyocarditis virus (ECMU) and capable of translating two open reading frames (OBEs) from one tRNA; plasmids derived from Escherichia coli11 (such as pSC4, pCP2. pCP2.1. pBB322, pBB325, pIS12 and pIS13); plasmids derived from Bacillus sylvius (such as pIV110, pTP5 and pC194); plasmids derived from yeast (such as p8H19 and p8H15); bacteriophages, such as λ phage; animal viruses such as retrovirus, smallpox vaccine virus (drastically lower) and baculovirus; pA1-11, pXT1, pBc / SMU, pBc / V8U, pCOXA1 / No. and so on. For the present invention, it is preferable to use the p1KE8 vector. With this vector, it is possible to prepare a replication cassette containing the “human telomerase promoter, E1A gene, zeidese sequence ΙΡΕ8 and E1B gene” in this order, by inserting the necessary genes into the multicloning site. For ligation of DNA acids, a DNA ligase can be used. Next, an example will be specifically described in which ETEWT is used as a human telomerase.

The E1A gene and E1B gene can be amplified in cells with expression of the E1 gene, such as 293 cells, by BT-RSV and / or ΟΝΛ-PCP using primers such as E1A-8, E1A-A8, E1B-8 and E1B- A8. If necessary, their sequences can be confirmed by a known method, such as TA cloning. Then, fragments E1A and E1B for DNA can be excised using a known restriction enzyme.

Then, it is possible to prepare a replication cassette consisting of HTEBT-E1A-1BE8-E1B for use in the present invention by introducing individual genes into a multicloning site and a similarly known vector (such as vector p1BE8), so that the following order is applied: “ETBT promoter sequence E1A -1KE8-E1B. " Or, if necessary, it is also possible to remove the cytomegalovirus (SMU) promoter from a known vector, such as p8yi1e, using known restriction enzymes and introduce into this site a sequence excised from pTEWT-E1A-1BE8-E1B using the corresponding restriction enzymes. Adenovirus for use in the present invention, with a single replication cassette consisting of YTEVT-E1A-1BE8-E1B, is called "Telomelysin." By expressing the E1 gene, which is necessary for the proliferation of adenovirus under the control of the HTBTpromotor, it is possible to proliferate the virus in a cancer-specific aspect.

In the recombinant virus used as a reagent in the present invention, the tagging cassette is also included with the replication cassette. A “tagging cassette” is included in the E3 region of the virus genome.

It should be noted here that the primary function of the viral vector used in the present invention is cytotoxicity during virus replication. Therefore, in order to use the reagent of the present invention in order to diagnose tissues with micro cancer, cytotoxicity must take place as late as possible. This is the case, since the fluorescence caused by the replication of the recombinant virus of the present invention disappears when the cells are destroyed and it becomes difficult to determine the location of the tissue affected by the micro cancer.

On the other hand, E3A and E3B exist in the E3 region of adenovirus and 11.6 kEA AER (adenovirus death protein) in the E3A region has the function of stimulating the cytotoxicity and spread of the virus.

Therefore, in the recombinant virus used according to the present invention, regions of the viral genome encoding proteins with the function of stimulating cytotoxicity and dispersion of the virus (such as the AIR-encoding region E3) are removed to thereby delay cell death and facilitate the determination of cancer cells based on SER emission fluorescence or the like.

The tagging protein that makes up the tagging cassette is a protein that glows in those cells in which the virus described above was replicated and visualized. It is preferable to use a substance that emits fluorescence. Examples of such substances include, but are not limited to, green fluorescent protein (CEP), obtained from luminous green jellyfish, such as Aediogea U1c1opa, CEP with increased luminescence (ECEP) or CEP with a shift to the red side (gCEP), which are modified variants of SER (SER uapap1z). It is also possible to use a yellow fluorescent protein (UEP), a cyan fluorescent protein (CEP), a blue fluorescent protein (BEP), or CEP obtained from Vepsha heshEogt1z. A gene encoding each of these proteins can be used in the present invention.

A promoter capable of regulating the expression of the above gene can be any promoter, since it is compatible with the virus used to express the above gene of interest. Specific examples of such promoters include, but are not limited to, the cytomegalovirus (SMU) promoter, the LTE promoter, the 8Y40 late promoter, the MMTU LTB promoter

- 5 011880 torr, K8U LK promoter and 8Ka promoter. Preferably, the SMC promoter or the 11TEKT promoter can be used.

The recombinant gene contained in the labeling cassette of the present invention can be obtained using conventional genetic engineering techniques. For example, a nucleic acid synthesis method using a commonly used DNA synthesizer can be used as a genetic engineering method. Another way - after the matrix is isolated or synthesized, primers specific for each gene can be developed according to the method, and the genetic sequence can be amplified using the CSC apparatus (CSC method; Siegep! Prgo! Oo18 ίη Mo1ci1ag Vu1odu, 1oBn ^ Uneu & 8op § (1987), 8syop 6.1-6.4); or you can apply the method of gene amplification using a cloning vector. With the usual skill in this area, it is easy to implement the above methods, being guided, for example, by Möläehlag Schlöpd 2 ' ¹ Eb. Purification of the obtained RAC product can be performed by known methods, such as the method with ethidium bromide, the method with 8 UVK green I (molecular probes), the method with ΟΕΝΕΟΈ-ΕΑΝ (E-pack), ΟΙΑΟΕΝ (ΟΙΑΟΕΝ), etc. using an agarose gel, method with IEAE-cellulose filter, freeze-squeeze method or dialysis tube method. When an agarose gel is used, the PCT products are subjected to agarose gel electrophoresis and the resulting DNA fragments are excised from the gel and purified. If necessary, it is possible to confirm by conventional sequencing methods that the expected gene has been obtained. For example, for this purpose, you can use the method of chain dideoxynucleotide termination (8apdeg e! A1. (1977) Proc. No. 111. Asab. 8cg I8A 74: 5463) or similar methods. Alternatively, you can also analyze the sequence with a suitable DNA sequencer (for example, ΑΒΙ PK18M; ArrPeb Vyukuyeshk). Further, the genes thus obtained are treated with known restriction enzymes. The recombinant gene is constructed in such a way that the resulting DNA fragment, the processing result (encoding a labeling protein) is located in the forward direction from the gene fragment encoding the promoter described above. Here you can apply as a plasmid bifunctional (shuttle) plasmid. Two genes (for labeling a protein and a promoter) are ligated using DNA ligase and introduced into the vector to thereby prepare a recombinant gene for the labeling cassette.

As a known vector, you can use the vector p8Li1e, a plasmid obtained from ExeptSoysoi (such as pCK4, pCK2, pCK2.1, pBK322, pBK325, pIS12 or pIS13); a plasmid derived from Bacillus pylori (such as pCV110, pTP5 or pC194); a plasmid derived from yeast (such as p8H19 or p8H15); bacteriophage, such as λ, phage; an animal virus such as a retrovirus, a vaccination virus or a baculovirus; pA1-11, pXT1, pKs / SMU, pKs / K8U, pSEI1 / Ieo or the like.

Further, a recombinant gene including the replication cassette and the labeling cassette described above is excised using the appropriate restriction enzymes and introduced into the corresponding viral expression vector to thereby prepare the recombinant virus. Examples of viral expression vectors include adenovirus, retrovirus, vaccine virus, and baculovirus. As described above, adenovirus (especially type 5 adenovirus) is preferred. Methods such as electroporation, liposome, spheroplast, and lithium acetate can be used to integrate the cassette into the virus.

In the present invention, in particular, a recombination gene can be prepared by introducing SMU-ECEP-8U40P (A) from RESE-I1 (CEOITESN) into the shuttle plasmid pHM11 and introducing the Skr451 fragment of this plasmid into the site C1a1 of the p8iyi1e vector into which ryTEKT E1A-1KE8-E1B.

In the present invention, in particular, the sequence of the desired region can be excised with restriction enzymes from the recombinant gene, as prepared above, and introduced into the virus DNA in the form of Abepo-X U1a1 DNA using a commercial kit such as Abepo-X Exrgeop 8u51esh (COOITES) (the resulting product is designated as AbepoX-11A1B).

This AbepoX-11A1B is linearized using a known restriction enzyme and then transferred to cultured cells, such as 293 cells, to thereby prepare an infectious recombinant virus.

The range of target cancer cells to be detected according to this invention is not limited. Any cancer cells can be used. For example, you can use solid types of cancer on the head and neck, in the stomach, colon, lung, liver, prostate, pancreas, esophagus, bladder, gall bladder / bile duct, breast, uterus, thyroid, ovary, etc. .; or leukemia, lymphoma, sarcoma, mesenchymal tumors or the like. Most cancer cells derived from human tissues exhibit an increase in telomerase activity. The present invention can detect such cancer cells in general where proliferation is activated by such telomerase activity.

Since the expression of telomerase in cancer cells is extremely high compared to normal cells, LTEKT is expressed in cancer cells containing telomerase and a replication cassette functions in them. As a result, the virus replicates, which in turn increases the replication of the marking protein. In this way, the tagging protein is expressed and visualized.

- 6 011880

Therefore, when the reagent of the present invention does not fluoresce in normal cells, it gives fluorescence in cancer cells. Thus, it becomes possible to visually observe cancer cells.

For infection of cells with a recombinant virus, for example, the following method can be used. First, cells, such as cancer cells 8 \ U620 of the human colon, cancer cells of the human lungs A549 and H1299, are applied to culture plates with the appropriate culture medium and cultured in the presence of CO ₂ gas at 37 ° C. As a culture medium, one can be used, which is usually used for culturing animal cells, for example, ΌΜΕΜ, MEM or или-1640. If necessary, serum, antibiotics, vitamins or similar substances can be added to it. Cultured cells are infected by plating a certain amount (0.1-10 ΜΟΙ (multiplicity of infection), preferably 1 ΜΟΙ) of the recombinant virus of the present invention. ΜΟΙ means the ratio between the amount of virus (infectious unit) and the number of cells when a certain number of cultured cells are infected with a certain number of virus particles. ΜΟΙ used as an indicator for infection of cells with the virus.

To confirm viral replication, virus infected cells extract and extract DNA from them. Then the DNA is subjected to CSC. in real time, using primers aimed at a specific gene located in the virus of this invention. Thus, a quantitative analysis is possible.

Regarding the identification of labeled cells: cancer cells can be seen because cells with virus replication emit specific fluorescence (for example, green light when CEP is used) when exposed to exciting light. For example, when cells infected with a virus are observed under a fluorescence microscope, it can be observed that the cells emit CER fluorescence. In order to observe infected cells over time, CEP fluorescence can be observed using an SSE (Microscopy Coi1go11eb Ecr1au) camera.

For real-time labeling and detection of the ίη νίνο cells of interest to us, the recombinant virus of the present invention can be introduced into a living organism.

The reagent of the present invention can be applied directly to the affected area. Another possibility is the reagent of the present invention can be introduced into a living organism (target cell or organ) by any known method, for example, by intravenous, intramuscular, intraabdominal or subcutaneous injection; inhalation through the nasal cavity, oral cavity or lungs; through the mouth; insertion into vessels with a catheter, etc. The dose level is chosen accordingly, depending on the active component, route of administration, target of administration, as well as age, body weight, gender, symptoms and other aspects of the patient's condition. Typically, the dose level can be chosen so that the virus of the present invention (active principle) is used as a daily dose of about 10 ⁶ -10 PFU (PEU-plaque forming units), preferably about 10 ⁹ '-10 ¹¹ PFU. This amount is given once a day or it can be divided into several portions and given several times a day.

The reagent of the present invention makes it possible to observe the label ίη νίνο in true time. Thus, the reagent of the present invention has the advantage of being used ίη νίνο diagnostic agent. This is useful in so-called navigational surgery.

If a wide excision is used, including the affected organ, during a surgical operation, a patient who has survived a surgical operation can expect a long life. However, the frequency of complications caused by the surgical operation itself remains high. Further, the loss of functioning of the excised organ inevitably affects everyday life after surgery. When treating cancer, it is important to use minimally invasive treatment, which will reduce the severity for the patient while maintaining long-term survival.

When the goal is to minimize the excision area, it is necessary to have information about the presence or absence of metastatic lymph nodes. As a method of obtaining such information, the “sentinel lymph node” (SU - 8Ν) attracts attention. SU is a lymph node that first receives lymph flow from tumors, and there is a hypothesis that the first micrometastases form in such a lymph node. This hypothesis is known as the theory of SU. Although large-scale clinical trials of breast cancer have already begun, first in Europe and the United States, it is not yet known whether this theory applies to other dense tumors. The tests have just begun.

The диагη νίνο cancer diagnosis system using the reagent of the present invention can lead to the development of a technology for the direct expression of fluorescent protein in cancer cells and the determination of tumor tissue or metastatic lymph nodes using a highly sensitive fluorescence detection system during surgery. In other words, it is possible to create the technology of “navigation surgery” as a more sensitive method than SU. The recombinant virus of the present invention replicates in a large number of cancer cells with telomerase activity, and these cells can emit, for example, strong green SER fluorescence.

When analyzing metastases in the lymph nodes, it was found that about 10% of the displaced metastases, i.e., incipient metastases in the second and subsequent nodes, report the onset of metastases in the second group or more distant lymph nodes, have moved from the lymph node

- 7 011880 of the first group that jumped over the first group of lymph nodes. Based on this report, many experts pointed out the danger of navigation SU. However, the cancer diagnosis system ίη νίνο using the reagent of the present invention identifies tumor tissues or metastatic lymph nodes directly during real-time surgery and navigation throughout the excision range. This system is original and creates an era, and in addition, it is extremely practical for a smooth operation. In particular, the reagent of the present invention is introduced endoscopically into the region of the tumor (for example, into the mucous membrane of the stomach or colon around the stomach cancer or colon cancer; into the inner region of tumors such as stomach cancer, colon cancer, lung cancer, pancreatic cancer) a few days before the surgery in the same manual way as when navigating SU. Then, sufficient time is needed for the virus to spread to tissues infiltrated by the tumor, metastatic tumor tissues or the accompanying lymph nodes, and replicate in the tumor sites or places positive for metastases.

During surgery, the light that stimulates ORP fluorescence is projected from the light source onto the surgical field after glancing and images from a special 3SSE camera are projected onto the display (a ас η))) у у у). Using a portable magnifier, you can find the field of view within the actual surgical field and it is possible to find lymph nodes that are positive for metastases on overlapping ORP images. Further, by installing a special filter, it is possible to distinguish fluorescence visually, without the use of a camera.

2. Reagent for the diagnosis of ex νίνο

The reagent of the present invention is also applicable as an agent for diagnosing ex νίνο for selection. In the current work, the quantification of tumor markers is the most common method for detecting the presence of cancer, which cannot be determined visually or its primary location cannot be determined.

However, tumor markers are not always satisfactory for specificity for cancer. In addition, it is extremely difficult to detect all types of cancer using a single marker.

It was shown that the telomerase activity of human malignant tumors increases by 85% or more and therefore its specificity for cancer is extremely high.

Diagnosis of ex νίνο cancer using the reagent of the present invention can be performed, for example, as described below.

Red blood cells are collected from a total blood sample taken from a patient. The reagent of the present invention is added to the remaining liquid with a suspension of cells in a certain ratio (0.1-10 ΜΟΙ, preferably 1 ΜΟΙ) and mixed in a test tube. The mixture is left for a certain time (for example, 12-48 hours) so that the cancer cells become infected with the virus and for subsequent replication of the virus. Then, OPP expression in the cell fraction is quantified by flow cytometry. Using this system, it becomes possible to detect free cancer cells present in peripheral blood with a high degree of sensitivity. This method can be used to detect free cancer cells present in peripheral blood in very small quantities.

3. Agent inducing cell death, and a method for inducing cell death

One of the results of the present invention is an agent that induces cell death, including a recombinant virus, in which a replication cassette with a human telomerase promoter, the E1A gene, the и8 sequence and the E1B gene are in this order included in the E1 division of the virus genome and the death inducing cassette, with a gene encoding a protein associated with the induction of cell death, and a promoter capable of regulating the expression of a gene encoding a protein are integrated into the E3 department of the virus genome. It is preferable to use the agent that induces cell death according to this invention in the gene therapy of cancer as an agent that induces the death of cancer cells, as well as for the prevention of relapse, inhibition and / or prophylaxis of metastases after surgery for cancer.

In the cassette inducing cell death of a recombinant virus contained in the cell death inducing agent of the present invention, a gene is integrated that operates on a promoter and which encodes a protein capable of inducing cell death.

This cell death inducing cassette used in the recombinant virus contains a gene encoding a protein associated with the induction of cell death and a promoter capable of regulating gene expression. Therefore, when the cell death inducing agent of the present invention is introduced into cancer cells, the virus replicates specifically in cancer cells. As a result, the level of expression within the cells of the protein associated with the induction of cell death increases and the induction of cell death occurs only in cancer cells without damage to normal cells.

A gene encoding a protein associated with the induction of cell death corresponds to a gene encoding a protein associated with the induction of cell death in a particular cell.

Particular examples of proteins associated with the induction of cell death include the following proteins, but are not limited to. In the present invention, a gene encoding any of

- 8 011880 of these proteins.

As a particular example of a protein associated with immunity, PA28 can be called. PA28 is a protein that activates intracellular proteasomes. When overexpressed, this protein causes immunological reactions and at the same time induces cell death. As a particular example of an apoptosis inducing protein, TKA1b can be called. TK1b is a molecule that induces the death of apoptotic cells by binding to a receptor on the cell surface. As a particular example of a protein associated with telomerase, AI5 can be called. AI5 has a sequence capable of inducing cell death of cells with telomerase activity.

The genes of these proteins associated with the induction of cell death can be obtained by conventional genetic engineering methods. For example, as a method of genetic engineering, you can call the method of nucleic acid synthesis using a commonly used DNA synthesizer. Another option, after the genetic sequence that is needed as a template is isolated or synthesized, primers specific for each gene can be developed, and the genetic sequence can be amplified using the CSC apparatus (CSC method; SiggeSh Prgo1oso1z ίη Mo1esi1ag B1o1odu, ίοΐιη \ UPeu & 8opz ( 1987), 8syop 6.1-6.4); or a gene amplification method using a cloning vector can be applied. In ordinary skill in this field easily perform the above methods, guided, e.g., Mo1esi1ag S1oshpd 2 ^'1 Eb., So1b 8rgshd Nagog aogaTogu Presse (1989). Purification of the obtained CSC products can be performed by known methods, such as the method with ethidium bromide, the method with 8ΥΒΚ green I (molecular probes), the method with ΟΕΝΕίΧΕΑΝ (Ripacoses), ΟΙΑΟΕΝ (ΟΙΑΟΕΝ), etc., using an agarose gel, the method using a filter from ΌΕΑΕ cellulose, freeze-squeeze method or dialysis tubing method. When an agarose gel is used, the CSC products are subjected to agarose gel electrophoresis and the resulting DNA fragments are excised from the gel and purified. If necessary, it is possible to confirm by conventional sequencing methods that the expected gene has been obtained. For example, for this purpose, you can apply the method of chain dideoxynucleotide termination (8apdeg e1 a1. (1977) Proc. No. 111. Asab. 8sk I8A 74: 5463) or similar methods. Another possibility is that you can also analyze the sequence with a suitable DNA sequencer (for example, AB1 PK18M; Arrieb WyuzuzTetz).

Anti-oncogenes are also among the substances that induce the death of cancer cells, since anti-oncogenes have the function of inhibiting the replication of cancer cells. For this purpose, the following anti-oncogenes used in conventional gene therapy can be listed:

p53 (8ЕО ΙΌ N0: 11; Asseszyup Jo. / deposit number / М14694): different types of cancer;

p15 (8E0 ΙΌ N0: 12; Assesiup Jo. L36844): different types of cancer; p16 (8E0 ΙΌ N0: 13; Assesiup Jo. L27271): various types of cancer;

ARS (8E0 ΙΌ N0: 14; Asseszyup Jo. M74088): colon cancer, stomach cancer, pancreatic cancer;

BKSA-1 (8Е0 ΙΌ N0: 15; Asseszyup No. Sh4680): ovarian cancer, breast cancer;

ERS-4 (8Е0 ΙΌ N0: 16; Asseszyup No. I44378): colon cancer, pancreatic cancer;

ΡΗΙΤ (8Е0 ΙΌ N0: 17; Asseszyup No. NM 112012): stomach cancer, lung cancer, uterine cancer;

p73 (8E0 ΙΌ N0: 18; Assesiup No. Υ11416): neuroblastoma;

RATSNEE (8Е0 ΙΌ N0: 19; Asseszyup No. I59464): basal cell carcinoma;

KPr 10 (8Е0 ΙΌ N0: 20; Asseszyup No. M1 5400): lung cancer, osteosarcoma;

OSS (8E0 ΙΌ N0: 21; Asseszyup No. X76132): colon cancer;

N61 (8E0 ΙΌ N0: 22; Assesup No. NM 000267): neurofibromatosis type 1;

N62 (8E0 ΙΌ N0: 23; Assesup No. L1 1353): neurofibromatosis type 2;

^ T-1 (8Е0 ΙΌ N0: 24; Asseszyup No. NM 000378): Wilms tumor.

These anti-oncogenes can be obtained by conventional genetic engineering methods. For example, a nucleic acid synthesis method using a conventional DNA synthesizer can be used as a genetic engineering method. Another option is that after a genetic sequence has been isolated or synthesized for use as a template, primers specific for each gene can be developed and the genetic sequence can be amplified using the CSC apparatus (CSC method); or a gene amplification method using a cloning vector can be used. If necessary, it is possible to confirm by conventional sequencing methods that the expected gene has been obtained.

As a promoter capable of regulating the expression of the above gene, any promoter can be used, since it is a suitable promoter compatible with the virus used to express the gene of interest to us. It is preferable to use the SMU promoter or the CTECT promoter. However, other promoters such as the 8Y40 late promoter, the MMTU bTK promoter, the K8U bTK promoter, and the 8Ka promoter can also be used.

The cell death inducing agent of the present invention can be applied directly to the affected area. Another option is the agent of the present invention can be introduced into a living organism (target cell or organ) by any known method, for example, by intravenous, intramuscular, intra-abdominal or subcutaneous injection; inhalation through the nasal cavity, oral cavity

- 9 011880 or lungs; through the mouth; through vessels with a catheter, etc.

The cell death inducing agent of the present invention can be applied, for example, by using methods such as freezing, so that it is easier to handle and then use individually or prepare pharmaceutical formulations by mixing with known pharmaceutical acceptable carriers such as excipients, filters, bandages, greases; or known additives (such as buffers, isotonic agents, chelating agents, colorants, preservatives, flavors, flavorings and sweeteners).

The cell death inducing agent of the present invention can be used orally or parenterally depending on the form of the agent, for example, orally in the form of tablets, powders, granules, pills, liquids, syrups and the like. and parenterally in the form of injections, externally in the form of suppositories, eye drops, and the like. Local injection into the muscles or abdomen or intravenous injection is preferred.

The dose level is chosen depending on the active ingredient, route of use, purpose of use, age, body weight, gender, symptoms and other aspects of the patient's condition. Typically, the dose level can be chosen so that the virus of the present invention (active ingredient) is taken as a daily dose of about 10 ⁶ -10 ¹¹ PFU (PFU - RBI, plaque forming units), preferably about 10 ⁹ -10 ¹¹ PFU. This amount is taken once a day or it can be divided into several portions and then it is taken several times a day.

When receiving the virus of the present invention, it is also possible to take a known immunosuppressant or analogue to suppress the immunity of a living organism and to facilitate infection with the virus.

Further, the virus of the present invention can be used together with at least one anticancer agent selected from the group consisting of known anticancer agents and radiation. Specific examples of anticancer agents include, but are not limited to, the following agents.

(1) Alkylating agents: these agents cause cytotoxicity by introducing alkyl groups into the nucleic acid / protein of cancer cells. Examples include carbocone, busulfan (mustard medicines) and mimustine (nitroureas).

(2) Antimetabolic agents: these agents have an effect that inhibits cell synthesis through the influence of antagonistic enzymes in metabolic processes. Examples include methotorexate (folates), mercaptopurine (purines), cytarabine (pyrimidines), fluorouracil, tegafur and karmofur.

(3) Antibiotics: these agents have anti-cancer effects. Examples include actinomycin Ό, bleomycin, adriamycin and mitomycin C.

(4) Anti-microtubule agents: these agents act on microtubules and exhibit anti-cancer effects. Examples include docetaxel, paclitaxel (taxanes) and vinorelbine, vincristine, vinblastine (alkaloids).

(5) Platinum preparations: these preparations inhibit DNA synthesis by forming cross bonds within or between DNA strands or DNA-protein cross-links. Examples include cisplatin, carboplatin and nedaplatin.

(6) Topoisomerase inhibitors: they include irinotecan (topoisomerase I inhibitor), podophyllotoxin derivatives (topoisomerase II inhibitor), etc. Topoisomerase is an enzyme that catalyzes the reaction of changing the number of DNA bonds by temporarily cutting off one or both DNA strands.

It is believed that the likelihood that the cell death inducing agent of the present invention will cause side effects is extremely small for the reasons described below. Thus, the cell death inducing agent of the present invention is a very safe preparation.

(1) The telomerase activity in normal cells is very small and the virus of the present invention is unlikely to be infectious in supporting cells such as hematopoietic cells.

(2) Since the virus of the present invention is capable of replication, it is possible to use this virus at a concentration lower than that of a conventional, non-replicating competent virus used in conventional gene therapy.

(3) Even when the virus of the present invention is given in excess, the antiviral effect occurs through the normal immune response of a living organism.

It is possible to induce cell death in the target cell by infection of the target cell with the recombinant virus of the present invention. The types of target cells are not particularly limited. For example, it is possible to use tumor cells, cells with active replication, cells whose telomerase activity is increased, etc.

“Infection of cells with a recombinant virus” has the meaning described above. To confirm whether or not cell death is induced, a morphological study can be performed as described below. Briefly, cells attached to the bottom of the culture plate are infected with the recombinant virus of the present invention. After a certain time, the shape of the cells becomes round, and they become suspended in the culture medium in the form of shiny cells that have detached from the bottom. To that

- 10 011880 time, the mechanism supporting the life of these cells is broken, and it can be noted that cell death is induced. Otherwise, it is also possible to confirm cell death using a commercial viable cell determinant kit using tetrazolium salts (e.g., MTT, XTT).

Below, the present invention will be described in more detail based on the following examples. However, the present invention is not limited to these examples.

Example 1. Visualization of cancer cells by co-infection ίη νίίτο.

In this example, it was previously clarified whether or not fluorescence can occur ίη νίίτο when the cancer cells are infected with the telomelysin virus containing the replication cassette and the non-replicating Ab-SER virus, including the marking cassette.

Human colon cancer cells 8ν620 and human lung cancer cells A549 and H1299 were infected with 0.1 ΜΟI (multiplicity of infection) from Ab-SER (Fig. 2).

As a result, the greening tendency was barely visible when human colon cancer cells 8 \ U620 and human lung cancer cells A549 and H1299 were infected with 0.1 ΜΟΙ (multiplicity of infection) from Ab-SER. However, when 1 ΜΟΙ of GCAE was used together, fluorescence could only be distinguished in cancer cells and no fluorescence was detected in normal cells such as human fibroblast cells \ UE38 and INP and human navel vascular endothelial cells (NIEC) (Fig. 3 )

Example 2. Visualization of cancerous tissues by co-infection ίη νίνο with telomelysin and Ab-SER.

In this example, it was previously ascertained whether ίη νίνο fluorescence would be present when the cancer tissues were infected with the telomelysin virus containing the replication cassette and the non-replicating Ab-SER virus containing the marking cassette.

Ab-SER (8x10 ⁵ PFU) and ΤΡΑΌ (8x10 ⁶ PFU) were introduced into the tumors of the human colon cancer 8 \ U5620 and the human lung cancer A549, previously transplanted to the dorsal side of hairless ("naked") mice. Then, fluorescence was observed after some time.

In all tumors, fluorescence spots began to be observed from the second day of administration and disappeared by the 14th day (Fig. 4).

Example 3. Detection of cancer cells using telomelysin-SER.

1. Preparation of expressing CEP, a replication competent virus (telomelysin CEP), which includes a replication cassette with a telomerase promoter and an E1 gene and a label cassette with a gene encoding CEP in a single virus.

The outline of telomelysin-SER is shown in FIG. 1. Telomelysin proliferates / replicates cancer cells specifically and telomerase specifically, since the E1L / 1KE8 / E1B is driven by an 11TEKT promoter. Further, telomelysin-SER also has a SER gene derived from Assciugs νίοΙοΓία, integrated into its E3 region, which is driven by a promoter. Therefore, cells in which virus replication is observed emit green fluorescence under exciting light, which allows the visualization of cancer cells.

Such a replication-competent virus was prepared as described below.

2. Preparation of a recombinant virus

From RNA extracted from 293 cells, an E1A gene of 897 bp was amplified. using CT-RSK, using specific primers (E1L-8 and E1L-L8) and RSK conditions described below.

E1A-8: 5’-ASA SSS OSA STO AAA ATS AS-3 ’(Zef GO N0: 5) E1A-A8: 5’-SAS ASO TTT ASA SST TAT SSS-3’ (Zef ΙϋΝΟ: 6)

The composition of the RAC solution: 1xRSC buffer

0.2 shM each 6ΝΤΡ3 tM M ^ CE

2.5 and AtrNTats Οοΐά

0.2 μΜ each primer

Reaction conditions: 95 ° С, 10 min (95 С, 1 min; 56 ’С, 1 min; 72” С, 1.5 min) x 32 cycles

C, 7 min

C, 5 min

From DNA extracted from 293 cells, the E1B gene from 1822 bp was amplified using ΌΝΑRSC using the following primers E1B-8 and E1B-A8.

The applied composition of the CSC solution and the reaction conditions (cycles, temperature) were the same as those used for amplification of the E1A gene.

Each RSK product was subjected to TA cloning (TA ί kit, Ίοηίηβ Κίΐ Fia1 RyuPuYu! ·: Ιηνί (το ^ η) to thereby confirm their sequence. Then DNA fragments from 911 bp (E1A) and

- 11 011880

1836 bp (E1B) were excised, respectively, using the restriction enzyme EcoKE

E1A and E1B were introduced into the M1i1 site and the 8a11 site, respectively, of the p1KE8 uss1og (COOXTESN) vector in the normal position (E1A-SHE8-E1B).

The 455 bp ETTEKT promoter sequence, which was cut using the restriction enzymes M1i1 and Bd111, was introduced into the X1yE site located in the opposite direction from E1A to E1A-SHE8-E1B with a normal orientation (pYTECT-E1A-1KE-E1B).

The cytomegalovirus (SMU) promoter contained in the p8th vector was removed by treatment with restriction enzymes M1c1 and Xe1. Then, a 3828 bp sequence excised from pYTEKT-E1A-1KE8-E1B using the restriction enzymes Xie1 and S0ΐΙ was introduced into this site (p8y-iA1B).

RESEAR-X1 (COOHTESN) was treated with Ade1 / Xue1, blunted with a Klenov fragment and self-ligated (RESE-X2).

This RESEAR-X2 was processed No. ίΙ / ΛΠΙΙ and blunted using T4 ECA polymerase, followed by the preparation of the Vd111 site using the Vd111 linker. This Vd111 fragment was introduced into the WatH1 site belonging to pHM11 (pHM11-ECEP-X2).

Next, the Czp451 fragment from pHM11-ECEP-X2 byd was introduced into the C1a1 site of the p8yiy1e vector into which pYTECT-E1A-1KE8-E1B was integrated (p8y-yA1B).

Sequence A 4381 bp was excised from the thus prepared recombinant gene (p8y-bA1B) using the restriction enzymes 1-Ce1 and P1-8ce1 and introduced into the Abepo-Χ viral DNA of the Abepo-Χ (СОХТЕСН) expression system (AbepoX-АА1В). This AbepoX-yA1B was treated with the restriction enzyme Ras1 for linearization and then transferred to 293 cells, thereby preparing an infectious recombinant adenovirus (hereinafter referred to as “telomelysin-SER”).

Example 4. Identification of human lung cancer cells.

1. Morphological changes in human lung cancer cells caused by infection with telomelysin-SER.

Cells extracted from non-small cell lung cancer, H1299, were cultured ίη ugo and infected with telomelysin-CER at a dose of 1 MO1 or 10 MO1. Specifically, H1299 cells were placed on 24-well plates, ⁵ x ^{10 4} cells per well. After 24 hours, the cells were counted and the virus was added in the culture medium to a concentration of 1 MO1 or 10 MO1. Then, the morphology of the cells was observed under a reversed microscope after a sufficient time that the cytotoxic activity of the virus was observed.

As a result, virus replication caused cell death depending on concentration and time. 120 h after infection with 10 MO1, the majority of the cells became round and when viewed under a reversed microscope, they were suspended (Fig. 5).

2. The emission of CER fluorescence by human lung cancer cells caused by infection with telomelysin-CER.

Images under a reversed (inverted) microscope shown in FIG. 6 was observed under a fluorescence microscope. Green CEP fluorescence was observed, indicating virus replication depending on concentration and time (FIG. 6). 72 h after infection with 10 MO1, CER expression was observed in the maximum number of cells. Then the number of SER-positive cells decreased and cell death was induced (Fig. 6).

3. Verification of telomelysin-SER replication based on quantitative CSC in real time.

H1299 human lung cancer cells were infected with telomelysin-CER at a concentration of 10 MO1. Cell samples were taken 2, 26, 50 and 74 hours after infection and DNA was extracted from them. Real-time CSCs were performed using the following primers targeting the Telomelysin-CER E1A gene to quantify the proliferation / replication of the virus. Primers and conditions applied are listed below.

E1A-8: 5’-SST OTO TOT AOA OAA TOS AA-3 ’(8EC U ΝΟ: 9)

E1A-A8: 5’-ASA OST SAA OTS SAA AOO TT-3 ’(8EC Ιϋ ΝΟ: 10)

The composition of the solution RAC :: 1xBC Ea5181ag1 ECA Maz1eg 8UVK green I tM M _ё C1 ₂

0.5 μΜ each primer

Keysop Sopbyups: 95 ° C, 10 min (95 ° C, 10 s; 60 ° C, 15 s; 72 C, 8 s) x 40 cycles

70'C, 15 _s

40’s, 30 s

The results showed that telomelysin-SER was already replicated 1,000,000 times 26 hours after infection (Fig. 7). Replication then reaches a plateau, but SER fluorescence is also slightly enhanced.

- 12 011880 after replication (Fig. 7).

Example 5. Detection of human colon cancer cells.

1. The emission of CER fluorescence of human colon cancer cells caused by infection with telomelysin-CER.

Cells extracted from human colon cancer, 8 ^ 620 were infected with telomelysin SER at 10 ΜΟΙ. Changes in the cells were observed over time under a reversed microscope and a fluorescence microscope.

As a result, CEP green fluorescence was detected, indicating virus replication versus time, as in the case of H1299 cells (Fig. 8).

2. Verification of telomelysin-SER replication using quantitative CSC. in real time.

In the same way as with H1299 cells, human colon cancer cells were infected with telomelysin-CER at 10 ΜΟΙ. Cell samples were taken at 2, 26, 50, 74 and 98 hours after infection and DNA was extracted from the bottom. Then virus replication was quantitatively analyzed by real-time CSC. Real-time CSCs were performed using the following primers directed to the telomelysin-CER gene E1A, so as to quantitatively analyze virus replication. Real-time CSC conditions (composition of the reaction solution, cycle, time period, etc.) were the same as for H1299 cells.

The results showed that telomelysin-SER was already replicated 1,000,000 times 26 hours after infection and almost reached a plateau 98 hours after infection (Fig. 9).

Example 6

1. Morphological changes in normal human lung fibroblast cells (INE) caused by infection with telomelysin-SER.

Normal human lung fibroblast cells (INE) were cultured ίη νίίτο and infected with telomelysin-CER at a level of 1 ΜΟΙ or 10 ΜΟΙ. Changes were observed under a reversed microscope for up to 120 hours after infection.

The result was that no morphological changes were noted and cell death was not induced (Fig. 10).

2. Emission of CER fluorescence by normal human lung fibroblast cells under the influence of telomelysin-CER infection

When the reversed microscope images shown in FIG. 10 was examined under a fluorescence microscope; emission of SER fluorescence was detected in some cells. However, taking into account the cell density, the emission was extremely weak compared to that emitted by cancer cells. Therefore, it is believed that telomelysin-SER hardly proliferates / replicates in normal cells (Fig. 11).

3. Verification of telomelysin-SER replication using quantitative CSC in real time.

The human lung cancer cell H1299, the human colon cancer cell 8 \ U620 and the normal human lung fibroblast cell (INE) were infected with telomelysin-CER at 10 ΜΟΙ as described above. Over time, cell samples were taken and DNA was extracted from them. Then quantitatively analyzed viral replication using CSC in real time.

The results showed that telomelysin-SER had already replicated about 1,000,000 times in cancer cells after 24 hours of infection and emitted noticeable SER fluorescence 72 hours after infection (Fig. 12). On the other hand, replication was only about 1000 times in YINYE cells even 72 hours after infection and CEP fluorescence was barely noticeable (Fig. 12).

Example 7. Identification of the proliferation / replication of telomelysin-CER inside the tumor using fluorescence imaging.

1. Telomelysin-SER (107 PFU) was injected into a human lung cancer tumor H1299 transplanted into hairless mice. Then, the emission of SER fluorescence was observed over time using an SSI camera.

The results showed that emission of SER fluorescence caused by telomelysin-SER replication could be detected 24 hours after infection and that the level of luminescence gradually increased 3 and 5 days after infection (Fig. 13).

2. In the same manner as described above, telomelysin-SER (10 ⁷ PFU) was injected into a human lung cancer tumor H1299 transplanted into hairless mice. After a week and three weeks later, the subcutaneous tumor was removed. The emission of CER fluorescence was observed on the whole tumor and on the incision using an SSI camera.

As a result, even when the fluorescence was weak on the surface of the removed tumor, telomelysin-SER replication could be observed over a wide range on the cut surface (Fig. 14). In the tissues three weeks after infection, fluorescence was visible over almost the entire surface of the tumor (Fig. 14).

3. In the same manner as described above, HT29 human colon cancer cells were transplanted.

- 13 011880 were injected into the rectal wall of hairless mice as an orthopic model and telomelysin CEP (10 ⁷ PFU) was introduced when a large tumor was formed. Emission of CER fluorescence caused by telomelysin-CEP replication was observed one week after infection with an SSI camera (Fig. 15). Fluorescence continued even three weeks after infection (FIG. 15).

Example 8. 1. Histological analysis of an orthorhopal model of rectal cancer using hairless mice and human colon cancer cells HT29.

The human colon cancer cell HT29 was transplanted into the rectal wall of the hairless mouse. When a large tumor formed, it was removed and studied after staining with hematoxylin-eosin (NOT).

As a result, a tumor formed around the rectum and a mass of tumor cells could be detected in the lymphatic vessels of the rectal wall (Fig. 16).

2. The results of the autopsy of an orthotopic model of colorectal cancer in hairless mice and human colon cancer cells HT29.

An HT29 human colon cancer cell was transplanted into the rectal wall and, when a large tumor formed, an autopsy was performed. As a result, swelling around the aorta was detected in three lymph nodes (ΕΝ) (Fig. 17).

3. Detection of intratumoral proliferation / replication of telomelysin-CER in an HT29 rectal tumor and lymph nodes near the aorta using fluorescence imaging.

The emission of CER fluorescence was detected on the basis of fluorescence imaging using an SSI camera in an HT29 transplanted rectal tumor and in one of three near-aortic lymph nodes (Fig. 18).

4. Detection of intratumoral proliferation / replication of telomilisin-CER in the near-aortic lymph nodes using fluorescence imaging.

Emission of SER fluorescence was detected based on fluorescence imaging using an SSI camera in only one of the three near-aortic lymph nodes (Fig. 19).

5. Detection of intratumoral proliferation / replication of telomilisin-CER in the near-aortic lymph nodes using fluorescence imaging.

Histological analysis of the near-aortic lymph nodes revealed metastatic tumor tissue in only one lymph node, which gave a positive reaction to CER fluorescence during fluorescence imaging using an SSI camera. Thus, it was confirmed that telomelysin-SER only replicates in lymph nodes positive for metastasis (Fig. 20).

Cited literature

Ke1b T., Sa1ash8 E., Lbts77e8e 1., 8ζθ I., Shli B.M., Lpbge \\ '8 1., Kapb1eu V., Ne18e S., Irpeiagb M., NaShe1b M., Koshe B., Kishb 1., Κίκη I. Neraye apepa1 sh1i8yup o e a ger11sa1yup-8e1eyeue opeu1u11e abepoupi (611520): rya8e II Ulga1, 1tipsyod1e, apb e11shea1 e-broch. Sapeig Ke8 62 (21): 6070-9, 2002.

Opportunity for industrial use.

In accordance with the present invention, a reagent for detecting cancer cells and an agent inducing cell death are provided. Since the reagent of the present invention can detect cancer cells with extremely high sensitivity even in a living organism, the reagent is useful in the field of so-called navigational surgery and the like.

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Claims (18)

CLAIM 1. A reagent for detecting cancer cells, characterized in that it includes a recombinant virus in which a replication cassette comprising a human telomerase promoter, E1 A gene, 1KE8 sequence and E1B gene in this order is integrated into the E1 region of the virus genome and the labeling cassette comprising a gene encoding a labeling protein and a promoter capable of regulating the expression of a gene encoding a labeling protein is integrated into the E3 region of the virus genome. 2. A reagent for the diagnosis of cancer, characterized in that it comprises a recombinant virus in which a replication cassette comprising a human telomerase promoter, E1 A gene, 1KE8 sequence and E1B gene in this order is integrated into the E1 region of the virus genome and the labeling cassette, comprising a gene encoding a labeling protein, and a promoter capable of regulating the expression of a gene encoding a labeling protein is integrated into the E3 region of the virus genome. 3. The reagent according to claims 1 and 2, characterized in that the reagent is used to detect or diagnose cancer ίη νίνο or for navigational surgery. 4. The reagent according to claims 1 and 2, characterized in that the promoter from human telomerase is an LTEKT promoter. 5. The reagent according to claims 1 and 2, characterized in that the labeling protein is a CBR. 6. The reagent according to claims 1 and 2, characterized in that the promoter capable of regulating the expression of a gene encoding a labeling protein is a cytomegalovirus promoter or LTEKpromotor. 7. The reagent according to claims 1 and 2, characterized in that the virus is an adenovirus. 8. An agent that induces cell death, characterized in that it comprises a recombinant virus in which a replication cassette containing a human telomerase promoter, E1A gene, 1KE8 sequence and E1B gene in that order is integrated into the E1 region of the virus genome, and the cassette, inducing cell death, including a gene encoding a protein associated with the induction of cell death, and a promoter capable of regulating the expression of a gene encoding a protein is integrated into the E3 region of the virus genome. 9. The cell death inducing agent of claim 8, wherein the human telomerase promoter is an LTEKT promoter. 10. The agent that induces cell death according to claim 8, characterized in that the protein associated with the induction of cell death is at least one of the proteins selected from the group consisting of a protein associated with immunity, a protein that induces apoptosis , and a protein associated with telomerase. 11. The cell death inducing agent of claim 10, wherein the protein associated with immunity is PA28. 12. The agent that induces cell death, according to claim 10, characterized in that the protein inducing apoptosis is a TKL1E. 13. The agent that induces cell death, according to claim 10, characterized in that the protein associated with telomerase is LI5. 14. The cell death inducing agent according to claim 8, characterized in that the promoter capable of regulating the gene encoding the protein associated with the induction of cell death is a cytomegalovirus promoter or LTEKT promoter. 15. The cell death inducing agent of claim 8, wherein the virus is an adenovirus. 16. The cell death inducing agent of claim 8, wherein the cell is a cancer cell. 17. A method for detecting cancer cells, characterized in that it includes infection of the cancer cells with a reagent according to any one of claims 1 and 3-6 and detecting fluorescence emitted by the cancer cells. 18. A method for inducing cell death in a target cell, characterized in that it involves infection of the target cell with a cell death inducing agent according to any one of claims 8-16.